Piezo2 voltage-block regulates mechanical pain sensitivity

Abstract PIEZO2 is a trimeric mechanically-gated ion channel expressed by most sensory neurons in the dorsal root ganglia. Mechanosensitive PIEZO2 channels are also genetically required for normal touch sensation in both mice and humans. We previously showed that PIEZO2 channels are also strongly modulated by membrane voltage. Specifically, it is only at very positive voltages that all channels are available for opening by mechanical force. Conversely, most PIEZO2 channels are blocked at normal negative resting membrane potentials. The physiological function of this unusual biophysical property of PIEZO2 channels, however, remained unknown. We characterized the biophysical properties of three PIEZO2 ion channel mutations at an evolutionarily conserved arginine (R2756). Using genome engineering in mice we generated Piezo2R2756H/R2756H and Piezo2R2756K/R2756K knock-in mice to characterize the physiological consequences of altering PIEZO2 voltage sensitivity in vivo. We measured endogenous mechanosensitive currents in sensory neurons isolated from the dorsal root ganglia and characterized mechanoreceptor and nociceptor function using electrophysiology. Mice were also assessed behaviourally and morphologically. Mutations at the conserved Arginine (R2756) dramatically changed the biophysical properties of the channel relieving voltage block and lowering mechanical thresholds for channel activation. Piezo2R2756H/R2756H and Piezo2R2756K/R2756K knock-in mice that were homozygous for gain-of-function mutations were viable and were tested for sensory changes. Surprisingly, mechanosensitive currents in nociceptors, neurons that detect noxious mechanical stimuli, were substantially sensitized in Piezo2 knock-in mice, but mechanosensitive currents in most mechanoreceptors that underlie touch sensation were only mildly affected by the same mutations. Single-unit electrophysiological recordings from sensory neurons innervating the glabrous skin revealed that rapidly-adapting mechanoreceptors that innervate Meissner's corpuscles exhibited slightly decreased mechanical thresholds in Piezo2 knock-in mice. Consistent with measurements of mechanically activated currents in isolated sensory neurons essentially all cutaneous nociceptors, both fast conducting Aδ-mechanonociceptors and unmyelinated C-fibre nociceptors were substantially more sensitive to mechanical stimuli and indeed acquired receptor properties similar to ultrasensitive touch receptors in Piezo2 knock-in mice. Mechanical stimuli also induced enhanced ongoing activity in cutaneous nociceptors in Piezo2 knock-in mice and hyper-sensitive PIEZO2 channels were sufficient alone to drive ongoing activity, even in isolated nociceptive neurons. Consistently, Piezo2 knock-in mice showed substantial behavioural hypersensitivity to noxious mechanical stimuli. Our data indicate that ongoing activity and sensitization of nociceptors, phenomena commonly found in human chronic pain syndromes, can be driven by relieving the voltage-block of PIEZO2 ion channels. Indeed, membrane depolarization caused by multiple noxious stimuli may sensitize nociceptors by relieving voltage-block of PIEZO2 channels.


Introduction
Piezo2 is genetically required for normal touch sensation (Chesler et al., 2016;Ranade et al., 2014;Coste et al., 2010Coste et al., , 2012, and it is widely assumed that PIEZO2 channels form the conduction pore of native mechanosensitive currents that underlie touch receptor mechanosensitivity. However, despite the fact that Piezo2 channels are expressed in almost all sensory neurons of the dorsal root ganglia (DRG), deletion of Piezo2 leads to complete loss of mechanosensitivity only in around half of all mechanoreceptors Murthy et al., 2018). Additionally, the mechanosensitivity of almost all nociceptors is preserved in the absence of Piezo2 (Murthy et al., 2018). Work in nematodes has shown how genetic deletion of candidate mechanotransduction channels does not always provide definitive evidence that the protein forms the pore of the native mechanosensitive current (Geffeney and Goodman, 2012;O'Hagan et al., 2005;Goodman and Sengupta, 2019). A powerful way to directly assess the participation of a channel in transduction is to change the biophysical properties of the endogenous channel with the prediction that native mechanosensitive currents should acquire these new biophysical properties (O'Hagan et al., 2005). PIEZO channels are not only gated by mechanical stimuli, but are also controlled by membrane voltage. Thus, at physiological membrane potentials >90% of PIEZO channels cannot be opened by mechanical stimuli, but are made available at depolarized membrane potentials (Moroni et al., 2018). We previously identified a single highly conserved Arginine residue in PIEZO1 channels (mR2482) that when mutated effectively eliminates most of the PIEZO1 voltage-block (Moroni et al., 2018) (Fig. 1a). Interestingly, mutations in the same conserved residue of PIEZO2 (mPIEZO2; R2756; hPIEZO2 R2686) are associated with distal arthrogryposis, Gordon syndrome and the Marden-Walker Syndrome, all of which are human developmental disorders (Alisch et al., 2016;Mcmillin et al., 2014). Here we show a single mutation can abrogate the voltage-block of the PIEZO2 channel, dramatically increasing channel availability at physiological membrane potentials. By mutating the same site in the channel in vivo we could investigate the effects of changing the channel properties on native mechanosensitive currents and their effects on sensory physiology. Surprisingly, we observed only minor effects on touch receptors, but the properties of mechanosensitive currents in nociceptors were dramatically sensitized in a way that reflected the changes in PIEZO2 channel function. Our data show how the voltage block of PIEZO2 serves to keep the mechanical threshold of nociceptors high so that they detect noxious and not non-noxious mechanical stimuli. Furthermore, our data suggest a simple model whereby different kinds of algogens drive nociceptor sensitization by releasing PIEZO2 channels from voltage-block.

Results
We first asked whether the conserved R2756 residue also controls the voltage sensitivity of mPIEZO2 channels. We thus generated mPiezo2 channels with single missense mutations (R2756H, R2756C and R2756K), known to be associated with human developmental diseases. We quantitatively assessed mechanosensitivity using substrate deflection of N2a Piezo1-/cells expressing wild type or mutant Piezo2 channels (Moroni et al., 2018;Poole et al., 2014;Servin-Vences et al., 2017) (Supplementary Fig. 1a-c). We measured three types of mechanically gated currents in cells expressing Piezo2 channels: rapidly adapting (RA), intermediate adapting (IA) and slowly adapting currents (SA) (Fig. 1b, Supplementary Fig.   1d). Cells expressing the R2756H, R2756C and R2756K mutations exhibited significantly fewer RA and increased proportions of IA and SA currents compared to wild type (Fig. 1b,   Supplementary Fig. 1d). The deflection-current relationship revealed that R2756K mutant channels are more sensitive to pilli deflection compared to wild type or R2756H/R2756C mutant channels (Fig. 1c, Supplementary Fig. 1e). Consistently the mean deflection threshold for R2756K was almost five-fold lower than that of wild type or R2756H/R2756C mutant channels (Fig. 1d, Supplementary Fig. 1f ). We also noted subtle, but significant changes in the kinetics of mechanosensitive currents generated by Piezo2 mutant channels (e.g. small increase in latency for activation) (Extended data Table 1). We next measured the effects of these mutations on the stretch and voltage sensitivity of PIEZO2 channels. Mutations were introduced into the stretch-sensitive chimeric channel mP1/mP2 (Moroni et al., 2018) and currents were measured from excised outside-out patches. The R2756K and R2756H mutant channels displayed significantly enhanced stretch sensitivity compared to wild type chimeric channels, but the R2756C substitution did not alter stretch sensitivity (Fig. 1e, Supplementary   Fig. 2a,b). Additionally, the R2756K and R2756H chimeric variants showed significantly slower inactivation kinetics compared to wild type channels ( Supplementary Fig. 2d). We next used a tail current protocol to measure channel availability (Moroni et al., 2018) (Fig. 1f).
Between 25 and 45% of the maximum tail current could be measured from the R2756H and R2756K chimeric variants at -60 mV compared to less than 5% in wild type ( Fig. 1f-g, Supplementary Fig. 3a). Thus, both R2756H and R2756K mutations substantially relieve the voltage-block of these chimeric channels at physiological membrane potentials. The effect on the tail current was not accompanied by any change in the rectification index ( Fig. 1h-i).
PIEZO2 channels inactivate very rapidly at negative potentials making it challenging to study deactivation kinetics. We thus measured the effects of pressure removal at a series of positive voltages on current deactivation and found that R2756H and R2756K chimeric variants showed significantly slower deactivation compared to the wild type chimera (Fig. 1j-k). A considerable delay in channel closing was also observed during the transition from the inactivated to deactivated state after pressure removal ( Supplementary Fig. 3b,d). In conclusion, slower inactivation and deactivation, increased mechanosensitivity and an almost complete removal of voltage-block were the main effects of the R2756H and R2756K mPiezo2 missense mutations, with the R2756K mutation clearly displaying the strongest effects on all these parameters. Our biophysical measurements led us to predict that introduction of R2756H and R2756K into the mouse genome should radically alter the mechanosensitivity of endogenous PIEZO2dependent currents. We generated two knock-in mice that globally express the R2756H and R2756K variants (Piezo2 R2756H and Piezo2 R2756K mice) (Fig. 2a, Supplementary Fig. 4a,b).
The orthologous human mutation of Piezo2 R2756H has been associated with short stature and scoliosis (Alisch et al., 2016;Haliloglu et al., 2017;Mcmillin et al., 2014). Interestingly, we found that homozygous Piezo2 R2756H/R2756H animals weighed on average ∼20% less than wild type controls at 4 weeks of age. At 8 and 12 weeks of age, both Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mice weighed significantly less on average than wild types (~9% less), however, this effect was only partially penetrant as many of the mutant mice had body weights in the same range as controls. No effect of the mutations on body weight were observed in heterozygous animals (Supplementary Fig. 4c,d). In 50% of the Piezo2 R2756K/R2756K mice (9/18) we observed abnormal spine curvature (scoliosis), but this phenotype was not observed in heterozygotes or in Piezo2 R2756H/R2756H mutant mice ( Supplementary Fig. 4e).
The introduction of missense mutations could alter gene expression, we thus examined Piezo2 expression in sensory neurons within the dorsal root ganglia (DRG) using RNAscope.
We found that in the DRG Piezo2 +/+ , Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mice showed similar Piezo2 mRNA levels (Fig. 2b, c). In the complete absence of Piezo2, around half of mechanoreceptors are completely insensitive to mechanical stimuli Murthy et al., 2018). We next recorded mechanosensitive currents in wild type and mutant sensory neurons in culture which had been classified as mechanoreceptors or nociceptors according to their size and action potential (AP) shape as previously reported (Lechner et al., 2009;Poole et al., 2014;Rose et al., 1986;Koerber et al., 1988) Table 2). We next recorded deflection gated currents from mechanoreceptors and again identified mechanically activated currents with RA, IA and SA kinetics, with RA-currents predominating (Poole et al., 2014;Hu and Lewin, 2006). Mechanoreceptors from Piezo2 R2756H/R2756H and Piezo2 +/R2756K showed a small but significant decrease in the proportion of RA currents compared to wild type cells, but no significant differences were observed in mechanoreceptors from Piezo2 R2756K/R2756K and Piezo2 +/R2756H mice ( Fig. 2e, Supplementary Fig. 6b). Deflection-current amplitude relationships were similar between genotypes with a trend for mechanoreceptors from Piezo2 R2756K/R2756K mice to show higher sensitivity ( Supplementary Fig. 6a,c). However, we observed robust and statistically significant reductions in the mean minimum deflection amplitudes capable of evoking mechanosensitive currents in all homozygous and heterozygous variant genotypes compared to wild type (Fig. 2f, Supplementary Fig. 6d). This change in threshold was accompanied by small changes in the kinetic parameters of mechanosensitive currents, for example in the inactivation kinetics of RA-currents in Piezo2 R2756H/R2756H mutants (Extended data Table 2).
We next asked if the threshold for gating the mechanosensitive currents in isolated sensory neurons was accompanied by changes in the properties of intact mechanoreceptors.
Using an ex-vivo preparation we recorded single mechanoreceptors innervating the hind paw glabrous skin (Schwaller et al., 2021;Walcher et al., 2018a). We found that rapidly-adapting mechanoreceptors (RAMs) from Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mutants that innervate Meissner's corpuscles displayed mildly enhanced firing to small 25 Hz sinusoidal stimuli compared to wild type (Fig. 2G). During the ramp phase of the mechanical stimulus RAMs recorded from Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K fired with shorter latencies reflecting lower force thresholds that were up to 10 mN smaller compared to wild type mice (~50% reduction) (Fig. 2h, Supplementary Fig. 7a,b). In contrast, slowly-adapting mechanoreceptors (SAMs) associated with Merkel cells Maksimovic et al., 2014) were barely affected by either missense mutation (Supplementary Fig. 7c-f). Thus, a sub-population of mechanoreceptors had significantly altered receptor properties when the biophysical properties of PIEZO2 are altered. This data is consistent with the idea that other unknown mechanosensitive channels regulate the sensitivity of many mechanoreceptors. We were surprised by the fact that large changes in the biophysical properties of endogenous PIEZO2 channels only had mild effects on touch receptors. However, there is increasing evidence that PIEZO2 may also play a role in pain sensitivity (Murthy et al., 2018;Szczot et al., 2018). Nociceptors that detect intense mechanical stimuli do not lose mechanosensitivity in the absence of PIEZO2, but show reduced initial firing to step mechanical stimuli (Murthy et al., 2018). Piezo2 is expressed by most nociceptors and so we next examined the effects of Piezo2 missense mutations on nociceptor physiology. We measured the mechanosensitivity of nociceptive sensory neurons with broad humped action potentials (Fig. 3a). We found that the deflection evoked currents were often three times larger at all deflection amplitudes in neurons from Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mice compared to wild type cells (Fig. 3b). In addition, the threshold for current activation was substantially lowered to values typical of mechanoreceptors in both types of mutant neurons (Fig. 3c, Fig. 2f). The frequency with which a mechanical stimulus evoked currents was also substantially increased in mutant neurons compared to wild type (Fig. 3d). We also noted significant, but much milder, increases in the sensitivity of deflection evoked currents in neurons from animals in which either mutation was present on only one allele ( Supplementary Fig. 8a-c). Normally, acutely cultured sensory neurons exhibit little or no ongoing action potential firing (Chakrabarti et al., 2020). Interestingly, we found that nociceptors from Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K often exhibited ongoing firing in the absence of current injection compared to wild type neurons (Fig. 3e,f). Moreover, we measured the rheobase of neurons from Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K animals and found this to be decreased by 30% and 55%, respectively compared to wild type ( Supplementary Fig. 9a,b). Such changes in electrical excitability could be due to alterations in voltage-gated conductance, however direct measurements of macroscopic voltage-gated inward and outward currents revealed no significant differences between wild type and mutant neurons ( Supplementary Fig. 9c,d ).
The resting membrane potential of mutant neurons was also not altered compared to wild type (Extended data Table 3). Thus, nociceptors from Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mice exhibit substantial mechanical hyperexcitability. We next examined intact nociceptors innervating the skin of which there are two main classes, thinly myelinated Aδ-fibers or unmyelinated C-fibers nociceptors (Lewin and Moshourab, 2004). In the glabrous skin we observed using quantitative force stimuli that C-fiber nociceptors from Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mice exhibited substantially increased firing rates and much lower mechanical thresholds for activation compared to wild type (Fig. 3g-j) as did Aδ-fiber mechanonociceptors ( Supplementary Fig. 10a-d). We analysed firing during stimulus onset (ramp phase) separately from the static phase and found that there was a substantial sensitization to both phases in C-fiber and Aδ-fiber mechanonociceptors in both mutant mice (Fig. 3i,j Supplementary Fig. 10 and 11). A hallmark feature of C-fiber nociceptors, first described by Perl in the 1960s, is that they often continue to fire after the noxious mechanical stimulus is removed (Bessou and Perl, 1969). Strikingly, C-fibers recorded from the mutants showed substantially increased ongoing firing after removal of mechanical stimuli compared to wild type C-fibers (Fig. 3g). We quantified this change in Cfibers from Piezo2 R2756H/R2756H , Piezo2 +/R2756K , and Piezo2 R2756K/R2756K mice and found that in these genotypes C-fibers exhibited up to three-fold increased interstimulus firing activity compared to wild type (Fig. 3k, Supplementary Fig. 11c). Only in Piezo2 +/R2756H mice, was the interstimulus firing was equivalent to that seen in wild type controls. These in-vitro and ex-vivo data show that voltage control of PIEZO2 channels in nociceptors is crucial for conferring high mechanical thresholds to mammalian nociceptors. Furthermore, the impairment in the ability of mutant channels to deactivate after opening was correlated with a large increase in ongoing activity of nociceptors in the absence of a mechanical stimulus. Apart from a non-penetrant scoliosis or occasional growth retardation, especially in Piezo2 R2756K/R2756K mice, the Piezo2 knock-in mice appeared largely healthy, with no obvious motor deficits. We tested behavioral responses to innocuous brushing of the hindpaw and found no obvious hypersensitivity in Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mice compared to wild type controls (Fig. 4a). However, paw withdrawal to punctate stimulation were clearly sensitized with paw withdrawal thresholds (PWT) (Chaplan et al., 1994;Christensen et al., 2020;Dixon, 1980) on average half those of controls in both Piezo2 R2756H/R2756H and Piezo2 R2756K/R2756K mutant genotypes (Fig 4c). Thus, relief of the voltage-block of Piezo2 is associated with heightened mechanical pain sensitivity in-vivo. Piezo2 R2756K/R2756K , n=9) and wild type (n=10) (c) Piezo2 R2756H/R2756H (n=19) and Piezo2 R2756K/R2756K (n=11) animals showed a reduced 50% PWT compared to controls (n=19) (One-way ANOVA test, **P=0.001). Each dot represents average values from different measures taken on different days in each animal. Data are presented as mean ± s.e.m.

Discussion
Here we have shown that the biophysical properties of PIEZO2 channels sets the sensitivity and mechanical thresholds of nociceptors required to detect painful mechanical stimuli.
Changing PIEZO2 residue R2756 to histidine or lysine made nociceptors approximately 3fold more sensitive to mechanical stimuli with mechanical thresholds similar to touch receptors. This remarkable change in excitability resembled physiological sensitization processes that follow strong chemical or mechanical activation of nociceptors in humans, primates and rodents (Lewin and Moshourab, 2004;Kress et al., 1992;Schmidt et al., 1995;Meyer et al., 1991;Steen et al., 1995). The observed changes in mechanosensitive currents measured from recombinant ion channels or isolated nociceptors were remarkably predictive of changes in the in-vivo sensitivity of nociceptors. For example, R2756 mutations dramatically slowed the closing of PIEZO2 channels a phenomenon that was reflected in ongoing activity of nociceptors after the cessation of the mechanical stimulus. We also show that R2756 mutations strongly influence the excitability of C-fiber nociceptors so that spontaneous activity was seen both in-vitro and in-vivo. This was a very surprising finding as it shows for the first time that it is not only voltage gated sodium channels like Na V 1.7, Na V 1.8 or Na V 1.9 that have the potential to control nociceptor excitability (Bennett et al., 2019), but also mechanosensitive channels that are controlled by membrane voltage.
Activation of nociceptors by inflammatory mediators or algogens, like capsaicin (Murthy et al., 2018), will strongly depolarize sensory endings thus potentially relieving voltage block of PIEZO2 channels. Membrane depolarization, which in the very compact nociceptor ending may be considerable, has thus the potential to mimic relief of voltage block that keeps the threshold to activate nociceptors high. Thus, we propose that voltage control of abundant PIEZO2 channels in most nociceptors is a major final mediator of nociceptor sensitization caused by strong nociceptive stimuli. In contrast to nociceptors, changing the biophysical properties of PIEZO2 was associated with only minor changes in the threshold and suprathreshold sensitivity of some, but not all touch receptors. The contrast between nociceptors and mechanoreceptors was striking and further supports the idea that uncharacterized mechanosensitive channels underlying touch sensation remain to be identified.

Methods
All experiments with mice were done in accordance with protocols reviewed and approved by the German Federal authorities (State of Berlin).

Molecular Biology
DNA constructs containing mPiezo2, mP1/mP2 and the variants were purified from
Negative masters were covered with polydimethylsilozane (PDMS, syligard 184 silicone elastomer kit, Dow Corning Corporation) mixed with a curing agent at 10:1 ratio (elastomeric base:curing agent) and incubated for 30 min. Glass coverslips were placed on the top of the negative masters containing PDMS and baked for 1h at 110° C. Pillar arrays were carefully peeled from the negative masters. The resulting radius-and length-size of individual pilus within the array was 1.79 µm and 5.8 µm, respectively. While the elasticity and the spring constant of each pilus was 2.1 MPa and 251 pN-nm, respectively, as previously reported (Patkunarajah et al., 2020;Poole et al., 2014;Servin-Vences et al., 2017). Before use for cell culture, pillar arrays were plasma cleaned with a Femto low-pressure plasma system (Deiner Electronic GmbH) and coated with EHS laminin (20 µg/mL) or Fibronectin from bovine serum (200 µg/mL). For pillar arrays experiments, a single pilus was deflected using a heat-polished borosilicate glass pipette (mechanical stimulator) driven by a MM3A micromanipulator (Kleindiek Nanotechnik, Germany) as described in Poole et al., 2014(Poole et al., 2014. NaOH (Moroni et al., 2018). Currents were recorded at 10 KHz and filtered at 3 KHz using an EPC-10 USB amplifier (HEKA, Elektornik GmbH, Germany) and Patchmaster software.

Ex-vivo skin nerve
Cutaneous sensory fiber recordings were performed using the ex-vivo skin nerve preparation.
Mice were euthanized by CO 2 inhalation for 2-4min followed by cervical dislocation. We used the recently described tibial nerve preparation to record from single-units innervating the glabrous hindpaw skin (Schwaller et al., 2021;Walcher et al., 2018a). In all preparations, the skin and nerve were dissected free and transferred to the recording chamber where muscle, bone and tendon tissues were removed from the skin to improve recording quality.
The recording chamber was perfused with a 32°C synthetic interstitial fluid (SIF buffer): 123 mM NaCl, 3.5 mM KCl, 0.7mM MgSO 4 , 1.7 mM NaH 2 PO 4 , 2.0 mM CaCl 2 , 9.5 mM sodium gluconate, 5.5 mM glucose, 7.5 mM sucrose and 10 mM HEPES (pH7.4). The skin was pinned out and stretched, such that the outside of the skin could be stimulated using stimulator probes. The peripheral nerve was fed through to an adjacent chamber in mineral oil, where fine filaments were teased from the nerve and placed on a silver wire recording electrode.
The receptive fields of individual mechanoreceptors were identified by mechanically probing the surface of the skin with a blunt glass rod or blunt forceps. Analog output from a Neurolog amplifier were filtered and digitized using the Powerlab 4/30 system and average amplitude 100 mN). Aβ-fibre slowly-adapting mechanoreceptors (SAMs) and rapidly-adapting mechanoreceptors (RAMs) were classified by the presence or absence of firing during the static phase of a ramp and hold stimulus, respectively as previously described (Milenkovic et al., 2008;Walcher et al., 2018b). Single-units were additionally stimulated with a series of five static mechanical stimuli with ramp and hold waveforms of increasing amplitude (3 s duration; ranging from ~10-160 mN). High threshold Aδ-and Cfibres were also stimulated using the five ramp and hold stimuli with increasing amplitudes.
Spontaneous activity in C-fibres was analysed after every mechanical stimuli.

Genotyping
Ear biopsies were collected and incubated overnight at 55° C while shaking at 800 rpm in a proteinase K-lysis buffer (200 mM NaCl, 100 mM Tris pH 8.5, 5 mM EDTA, 0.2% of SDS).
PCRs were performed using supernatant of the lysis preparation as DNA template (20-100 ng), 1X Taq PCR buffer, 2 mM MgCl 2 , 400 µM dNTPs, 1.25 U Taq-polymerase (Thermofisher Scientific) and 0.5 µM of primers. A 499 bp fragment of Piezo2 locus was amplified using the forward 5'-GAAAGAGCTACTTTGAAAGGAGTATGTGC-3' and reverse 5'-CCTGTCAGAAGAGAAATGGTTGCC-3' primers. Inserted point mutations generated new restrictions sites that allow to identify wild type, heterozygous and homozygous animals from each knock-in mice. PCR products were incubated overnight with BspI and MseI restriction endonucleases (NEB Inc.) for Piezo2 R2756H and Piezo2 R2756K mouse lines, respectively. Amplified and digested DNA fragments were observed by gel electrophoresis.

RNAscope
Lumbar DRGs were collected from adult animals and incubated for 40 min in Zamboni's fixative media (2% of para-formaldehyde + picric-acid), washed with PBS and incubated in 30% sucrose (in PBS) overnight at 4°C. DRGs were embedded in OCT Tissue Tek (Sakura, Alphen aan den Rijn). 10 µm-thick cryosections were stored at -80°C until used for experiments. In situ hybridization was carried out according to the manufacturer's instructions (RNAscope TM Multiplex Fluorescent V2 assay, ADC, Kit #323110, Piezo2 probe #4001191). LSM700 Carls Zeiss confocal microscope was use to acquired images at 20X and numerical aperture 0.5. Fluorescence intensity was analysed using ImageJ.

Brush and von Frey experiments
For both methods, testing was carried out during the light phase. Males and females were included in the experiments. Mice (7-12 weeks old) were placed in plastic cages with a metal grid floor bottom that allowed access to hindpaw stimulation. Animals were habituated for two-consecutives days for 20 min before testing. Before starting the behavioral test, animals were placed in the cages for at least 20 min (accommodation) and experimentation started when animals stopped cage exploration. Each animal was tested at least twice on different days and average values from those measures were plotted in each animal.
Responses to gentle touch was measured by stroking the surface of the hindpaw. Animals were stimulated five times with at least 2 min between each stimulation. The percentage was calculated according to the number of withdrawals out of the five stimulations.

Statistical analysis
All data analyses were performed using GraphPad Prism and all data sets were tested for normality. Parametric data sets were compared using a two-tailed, Student's t-test.
Nonparametric data sets were compared using a Mann-Whitney test. To compare more than two groups, One-way ANOVA was used. Categorical data were compared using χ 2 tests.